My long-term research objectives are to identify factors that play important roles in transcription by RNA polymerase II (Pol II) in vivo and to determine the mechanisms by which these factors act. The proposed studies reflect my longstanding interests in gene regulation and commitment to biomedical research. The experiments will be performed in a highly interactive and supportive research environment, and my progress will be greatly aided by institutional commitments to alleviate teaching and committee responsibilities. My immediate research goals center on transcription initiation and early post- initiation events. The Specific Aims represent extensions of our previous studies on Saccharomyces cerevisiae TATA box-binding protein (TBP). TBP binds directly to the TATA box at Pol II promoters and initiates a cascade of events that culminate in an RNA message. Therefore, an understanding of the factors that control TBP is a critical step toward understanding the regulation of gene expression in eukaryotes. Specific Aim 1 is to investigate the function of Rtf1, a novel protein important both for TATA site selection by TBP and for transcript elongation. Rtf1 and associated proteins will be purified, Rtf1- regulated genes will be identified using DNA microarrays, and genetic selections will be performed to identify suppressors of rtf1 mutations. In addition, the phosphorylation state of Pol II, which correlates with the transcription cycle, will be analyzed in various mutant strains. Specific Aim 2 is to elucidate the importance of TBP as a target for regulatory factors in vivo. Chromatin immunoprecipitation and in vivo footprinting methods will be used to determine the effect of gene-specific and globally acting transcription factors on the recruitment of TBP to a highly regulated model promoter. Specific Aim 3 is to analyze two distinct classes of TBP mutants and gain insights into two fundamental aspects of Pol II transcription: orientation specific assembly of the transcription complex and regulation of initiation. TBP mutants that exhibit reversed DNA binding polarity in vivo will be analyzed using DNA cleavage and transcription assays. TBP mutants that are altered in a particular subdomain of TBP and exhibit phenotypes indicative of transcriptional defects will be studied. The results from this work will provide a solid foundation for the continued dissection of transcription initiation and elongation in future years. Since the proteins and mechanisms employed in the regulation of Pol II are highly conserved, the information that is learned from these studies in yeast will significantly advance our understanding of transcription in humans, where an alteration in this process leads to important human diseases including AIDS.